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Journal of Lipid Research, Vol. 45, 2339-2344, December 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Validation of deuterium-labeled fatty acids for the measurement of dietary fat oxidation during physical activity

Aarthi Raman^*, Stephane Blanc, Alexandra Adams and Dale A. Schoeller^1,*

^* Interdepartmental Program in Nutritional Sciences, University of Wisconsin-Madison, Madison, WI
d'Ecologie et Physiologie, Strasbourg, France
Department of Family Medicine, University of Wisconsin-Madison, Madison, WI

Published, JLR Papers in Press, September 1, 2004. DOI 10.1194/jlr.M400289-JLR200

¹ To whom correspondence should be addressed. e-mail: dschoell{at}nutrisci.wisc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Measurement of ¹³C-labeled fatty acid oxidation is hindered by the need for acetate correction, measurement of the rate of CO₂ production in a controlled environment, and frequent collection of breath samples. The use of deuterium-labeled fatty acids may overcome these limitations. Herein, d₃₁-palmitate was validated against [1-¹³C]palmitate during exercise. Thirteen subjects with body mass index of 22.9 ± 3 kg/m² and body fat of 19.6 ± 11% were subjected to 2 or 4 h of exercise at 25% maximum volume oxygen consumption (VO_2max). The d₃₁-palmitate and [1-¹³C] palmitate were given orally in a liquid meal at breakfast. The d₃-acetate and [1-¹³C]acetate were given during another visit for acetate sequestration correction. Recovery of d₃₁-palmitate in urine at 9 h after dose was compared with [1-¹³C] palmitate recovery in breath. Cumulative recovery of d₃₁-palmitate was 10.6 ± 3% and that of [1-¹³C]palmitate was 5.6 ± 2%. The d₃-acetate and [1-¹³C]acetate recoveries were 85 ± 4% and 54 ± 4%, respectively. When [1-¹³C]acetate recovery was used to correct ¹³C data, the average recovery differences were 0.4 ± 3%. Uncorrected d₃₁-palmitate and acetate-corrected [1-¹³C]palmitate were well correlated (y = 0.96x + 0; P < 0.0001) when used to measure fatty acid oxidation during exercise.

Thus, d₃₁-palmitate can be used in outpatient settings as it eliminates the need for acetate correction and frequent sampling.

Supplementary key words substrate utilization • mass spectrometry • stable isotopes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Stable isotopes have been used to quantify plasma and dietary fat oxidation in the past (1–3). When a ¹³C-labeled fatty acid is dosed orally or by constant infusion, the ¹³CO₂ in breath can be used to measure the tracer oxidized. The ¹³C liberated during oxidative metabolism, however, is partly sequestered in the intermediates of the tricarboxylic acid (TCA) cycle (40%) and the bicarbonate pool (10%), causing fat oxidation to be underestimated. To correct for this sequestration, an additional dose of [1-¹³C]acetate is administered. Acetate is converted to acetyl-CoA and is oxidized in the TCA cycle (4). Unfortunately, sequestration is variable and depends on the conditions under which it is measured; hence, estimation of acetate sequestration is essential (5). In addition, the use of ¹³C-labeled fatty acid is further constrained by the need for frequent sampling of breath and the use of a metabolic cart or respiratory chamber to quantify the flux of CO₂ to calculate ¹³C recovery accurately. These factors increase subject burden; thus, an alternative method to quantify fat oxidation in the body would be useful.

One such method for the measurement of fat oxidation is the use of deuterium-labeled fatty acids (3, 6). When oxidized, ²H-labeled fatty acid is metabolized to acetyl-CoA, releasing NADH molecules. The ²H label is released as water, in part, when NADH molecules are oxidized in the respiratory chain. Oxidation of acetyl-CoA in the TCA cycle releases the rest of the deuterium label in the form of ²H-labeled water. This ²H₂O mixes with the body water and can be sampled in the urine (7). Urinary and insensible water losses are minimal (8); hence, the enrichment of label in urine can be used effectively to calculate the cumulative recovery of the label and hence the fat oxidized. Consequently, the need for measurement of CO₂ and flux is also eliminated.

Deuterium-labeled palmitic acid has been validated against acetate-corrected ¹³C-labeled palmitic acid during rest in humans by Votruba, Zeddun, and Schoeller (6), who dosed subjects at rest with ¹³C- and d₃₁-labeled palmitic acid and demonstrated that the cumulative recoveries for both tracers were highly correlated (y = 1.045x – 0.47; r ² = 0.88; P < 0.0002). More importantly, the mean difference in percentage recovery of the labels was 0.5 ± 2.8% when ¹³C data were corrected for acetate fixation. This method raises interesting possibilities for use under free-living conditions; however, this method has not been validated under nonresting conditions. Herein, we compared the metabolic fate of orally ingested d_31-palmitate and [1-¹³C] palmitate during physical exercise of varying durations at moderate intensity. The objective was to ensure the validity of ²H-labeled fatty acids as an accurate tool to measure dietary fat oxidation under a range of free-living conditions.

	METHODS

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Materials
Labeled fatty acids were obtained from Cambridge Isotope Laboratories (Andover, MA). [1-¹³C]palmitic acid and Na salt of [1-¹³C]acetate were 99 atom% ¹³C. The d₃₁-palmitic acid was 98 atom%, and Na salt of d₃-acetate was 99 atom% ²H. The ¹⁸O was obtained as water (Isotec, Inc., Miamisburg, OH) and was 10.8 atom%.

Subjects
Healthy volunteers (six males and six females) were recruited, and their characteristics are summarized in Table 1. Two additional subjects were recruited for the measurement of natural isotope abundances and daily natural variability of isotopes in the body (body mass index = 22.8 ± 3.2 kg/m²; mean ± SD). The experimental protocol was thoroughly explained to the subjects before their recruitment into the study, and a signed informed consent was obtained from each of the subjects. The protocol was approved by the Institutional Review Board at the University of Wisconsin, Madison. Subjects completed a Physical Activity Readiness Questionnaire (9) to screen out those at risk during exercise. The physical stress test [maximum volume oxygen consumption (VO_2max)] was used to determine their maximal oxygen-consuming capacities, and persons with abnormal cardiac output during this test were excluded from the study. Exclusion criteria included pregnancy; metabolic diseases such as diabetes, thyroid dysfunction, kidney malfunction, and cardiovascular diseases; and subjects who were advised by their physicians to not undertake any physical activity.

The 24 h energy requirements of the subjects were calculated from the World Health Organization (WHO) equation (10) and were divided into 40% at dinner, 30% at breakfast, and 30% at lunch. An activity factor of 1.5 for the previous day and 1.7 for the test day were added to the WHO calculated energy expenditure. The macronutrient distribution of each standard meal consisted of 50% carbohydrates, 35% fat, and 15% protein.

On the day of the test, subjects were awakened at 6:30 AM, and a urine sample was collected at 6:45 AM. Urine and breath samples were collected at 8:00 AM to measure the natural abundance of ²H in the body water and the natural ¹³CO₂ abundance in breath, respectively. The first visit consisted of an oral load of [1-¹³C]acetate at 2 mg/kg body weight and d₃-acetate at 10 mg/kg body weight mixed in a liquid replacement meal (Boost high protein; Mead Johnson Nutritionals) during breakfast. The second stay consisted of an oral load of [1-¹³C]palmitate at 10 mg/kg body weight and d₃₁-palmitate at 15 mg/kg body weight, mixed in the same liquid replacement meal during breakfast.

Subjects exercised at 10:00 AM for 2 h (2 hr-eEx) or 4 h at a light intensity (25% VO_2max) on a cycle ergometer. Within the 4 h exercise group (n = 8), three subjects started exercise at 10:00 AM [45 min after dose; early exercise (4 hr-eEx)] and five subjects started exercise at 1:00 PM [3 h, 45 min after dose; late exercise (4 hr-lEx)].

Measurements of CO₂ flux were taken for 20 min every hour. Respiratory gas exchange (RGE) was measured for 20 min every hour during the rest of the stay including at rest, during exercise, and after exercise using a Deltatrac I metabolic cart (Sensormedics). The O₂ and CO₂ analyzers were calibrated with a standard gas containing a 96% O₂ and 4% CO₂ mixture. The subjects breathed through a mouthpiece with their noses clipped to ensure complete respiratory gas monitoring. The valve is connected to a canopy system, which acts as a mixing chamber, and is exhausted into the Deltatrac system to measure the rate of oxygen consumption and carbon dioxide production. The first 5 min of measurement was excluded, and the hourly RGE was calculated assuming that the subsequent 15 min measurements per hour were representative of the whole hour.

[1-¹³C]palmitate oxidation rates were calculated using hourly breath samples collected when subjects blew through straws into 15 ml additive-free Vacutainers^TM (BD, Franklin Lakes, NJ), and d_31-palmitate oxidation rates were calculated from the spot urine samples collected every hour and stored in cryogenically stable tubes (Corning, Inc.). The recovery correction factor for [1-¹³C] palmitate and palmitate was determined using the [1-¹³C]acetate and d₃-acetate recovery rates.

Sample analysis
To measure the ratio of ¹³CO₂ to ¹²CO₂ in breath, a sample of breath was injected into a continuous-flow isotope ratio mass spectrometer (IRMS; Delta S; Finnigan MAT). The sample was injected into a continuous helium stream onto a Chromosorb-Q for separation of CO₂, which is directed into the source of the IRMS. Injections were made in duplicate, and the average standard deviation was 0.3ç. Postdose enrichments within a subject were calculated using baseline predose breath samples (7).

To measure the ratio of ²H to ¹H in urine, a sample (5 ml) of urine was mixed with 200 mg of carbon black to reduce impurities and was passed through a 0.45 µm filter (11). One milliliter of decolorized urine was placed in a 3 ml autosampler vial and analyzed for ²H/¹H ratios using the Delta plus IRMS (Finnigan MAT). A 0.8 µl aliquot was injected into a chromium-packed quartz tube held at 850°C to reduce water to hydrogen gas. Each sample was injected three times with independent analysis. Data were corrected for H³⁺ and memory errors. Results were corrected to the standard mean ocean water (SMOW) scale.

Total body water was estimated from ¹⁸O enrichments in urine samples collected at baseline and 4 h after dosing with ¹⁸O-labeled water. One milliliter of decolorized urine sample was allowed to equilibrate with CO₂ at 25°C for 48 h in a water bath. The ¹⁸O enrichment was measured using a continuous-flow IRMS as detailed by Schoeller and Luke (12). Total body water was calculated from the ¹⁸O data using the dilution method as described by Schoeller and van Santen (8).

Label calculations
²H and ¹³C label recoveries were calculated every hour for 9 h after dose. Baseline enrichment of the individual subject was subtracted from hourly recoveries to derive the increase in label recovery above baseline. Two subjects were made to follow the protocol without dosing, and the average enrichments per hour of these subjects were used to correct for natural variations in abundance of the isotopes attributable to meals and exercise. Thus, the final recovery values () represent individual recoveries per hour corrected for baseline abundance of individual subjects and for natural variation during the day. Rate of CO₂ production (V_CO2) was obtained from RGE measurements (6).

where V_CO2 is measured in milliliters per minute; R_STD = ¹³C/¹²C of standard CO₂; D = dose in grams; P = ¹³C isotope atom%; n = number of labeled atoms per molecule of tracer; MW = molecular weight of the tracer ([1-¹³C]acetate Na salt = 83; [1-¹³C]palmitic acid = 257); and isotopic enrichment above the baseline (Del) per mille () = (R_U/ R_STD – 1) x 1,000.

Breath samples were collected for 9 h after dose on both visits to correct for hourly acetate sequestration of acetate label in the TCA cycle. Deuterium recovery was calculated assuming that it is equally distributed across the total body water (TBW).

where TBW (moles) is multiplied by a factor of 1.035 to get a ²H dilution space from the TBW obtained by ¹⁸O dilution space; R_STD = ²H/¹H of SMOW; D = dose in grams; P = ²H isotope atom%; n = number of labeled atoms per molecule of tracer; MW = molecular weight of the tracer (d₃-acetate Na salt = 83; d₃₁-palmitic acid = 287). The hours after dose represents the midpoint between voids for ²H recovery calculations.

Because the mean difference in body weight between visits was 0.39 ± 1.3 kg, the TBW in these subjects was assumed to be constant.

Statistical analysis
A Student's paired t-test was used to compare both tracers. Regression analysis was done to correlate the two tracers. ANOVA was used to identify the effects of exercise on the correlation of the tracers. P 0.05 was required for statistical significance. All values are presented as means ± SD. All statistical analyses were performed using STATVIEW version 5.0.1 (SAS Institute, Inc., Cary, NC).

	RESULTS

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ANOVAs of the ratios of [1-¹³C]palmitate to d₃₁-palmitate recoveries did not show a significant difference between exercise groups (mean difference = 0.03%; P = 0.9). Hence, exercise groups were combined for this analysis.

Acetate recovery
Cumulative ¹³CO₂ recovery from [1-¹³C]acetate plateaued at 6 h after dose in all exercise groups, as shown in Fig. 1 . Cumulative [¹³C]acetate recoveries 3, 6, and 9 h after dose were 36.3 ± 9%, 52.6 ± 8%, and 56.8 ± 9%, respectively, indicating that most of the label was recovered at 6 h after dose in all exercise groups. ²H₂O recovery from d₃-acetate peaked at 45 min after dose in all three exercise groups, as shown in Fig. 1. Cumulative d₃-acetate recoveries 3, 6, and 9 h after dose were 92.1 ± 8%, 90.6 ± 4%, and 88.4 ± 4%, respectively, indicating that most of the label was recovered at 3 h after dose. Compared with [1-¹³C]acetate, d₃-acetate showed greater cumulative recovery (mean difference = 31%; P < 0.005) (Table 2). Instantaneous d₃-acetate recovery was significantly higher than ¹³C at any time during the measurement period.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Recovery of [1-13C]acetate and [2H]acetate expressed as a percentage of dose per hour (n = 12). Hydrogen recoveries were calculated as the cumulative recovery at each time point based on the increment in enrichment above the previous urine sample. The mean [2H]acetate recovery was 86 ± 4% (mean ± SD), and the mean [13C]acetate recovery was 57 ± 8.5% (mean ± SD). Deuterium recovery was significantly higher than 13C recovery for all exercise groups. 2hr-eE, 2 h early exercise; 4hr-eE, 4 h early exercise; 4hr-lE, 4 h late exercise. Closed symbols represent 2H acetate recoveries and open symbols represent 13C acetate recoveries.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Recovery of [1-13C]palmitate and [2H]palmitate expressed as a percentage of dose per hour (n = 12) uncorrected for acetate sequestration. Hydrogen recoveries were calculated as the cumulative recovery at each time point based on the increment in enrichment above the previous urine sample. The mean [2H]palmitate recovery was 10.3 ± 2.8% in all exercise groups together, and that of [13C]palmitate was 5.8 ± 1.8% in all exercise groups together. Uncorrected [13C]palmitate recovery was significantly different from uncorrected [2H]palmitate recovery (mean difference = 4.7%; P < 0.0001). Closed symbols represent 2H palmitate recoveries and open symbols represent 13C palmitate recoveries.

Validation
Correlation analysis showed that cumulative d₃₁-palmitate recovery uncorrected for acetate fixation was correlated with [1-¹³C]palmitate recovery group corrected for [1-¹³C]acetate fixation (r ² = 0.51; y = 0.55x + 4.6; SEM = 0.19; P < 0.02) (Fig. 3) . Because the y-intercept was not significant (P = 0.58), it was removed from the regression model (r ² = 0.2; y = 0.96x; SEM = 0.07; P < 0.0001). When d₃₁-palmitate recovery was corrected for acetate sequestration using group average d₃-acetate recovery, the correlation r ² was 0.56 (y = 0.74x + 4.6), and when an individual subject's d₃-acetate recovery was used to correct his/her own d₃₁-palmitate recoveries, the correlation r ² was 0.54 (y = 0.69x + 4.9). The intercept in both of these instances was significantly different from zero (P = 0.02). The mean difference between the two tracers was lowest when uncorrected d₃₁-palmitate recoveries were used (mean difference = 0.4 ± 3%; P = 0.64).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Group acetate corrected [2H]- and [1-13C]palmitate percent recovery at 9 h after dose during exercise. Acetate recovery averaged for the exercise group was used to correct for label sequestration. When [2H]palmitate was correlated with [1-13C]palmitate recovery group corrected for acetate, the correlation r 2 was 0.51 (y = 0.55x + 4.6; SEM = 0.19; P < 0.02).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Comparison of the average and difference between [2H]palmitate percent recovery and group acetate corrected [1-13C]palmitate percent recovery at 9 h after dose during exercise and rest. The mean difference between the two tracers was 0.03 ± 2.6% in the exercise group and –0.22 ± 2.6% when the rest data were included. The residuals for the combined data showed a normal distribution.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Although this validation was performed using stable isotope-labeled fatty acids, radioisotope-labeled fatty acids have also been used to measure dietary fatty acid oxidation under rest and exercise conditions. Romanski, Nelson, and Jensen (13) dosed their subjects with [³H]triolein and [¹⁴C]triolein along with their meal to study the trafficking of dietary fat toward oxidation or storage for 24 h after dose. Although acetate corrections for both radioisotopes were not measured, the cumulative ³H and ¹⁴C recoveries were correlated; thus, our results should apply equally well to these radioisotopes.

Carbon labeling of fatty acids appears as a natural choice for the measurement of oxidative processes, but it is subject to sequestration in the TCA, leading to reduced yield (14). Since Sidossis et al. (4) proposed the use of a correction factor for acetate sequestration in the TCA cycle intermediates, ¹³C-labeled fatty acids have been used for the measurement of substrate metabolism, including fatty acid oxidation rates, but their accuracy has been debated. Acetate exits the path of the labeled substrate metabolism before entry into the TCA cycle and hence can account for label sequestration occurring in the TCA cycle. The accuracy of correction for sequestration depends on the position of the label in the fatty acid and the physiological conditions under which its metabolism is measured (15).

Unlike labeled carbons, most of the ²H label from fatty acids is rapidly released as reducing power (NADH-H⁺ and FADH₂) during ß-oxidation and then again in the TCA cycle (4). Thereafter, the ²H is oxidized to water and released into the total body water pool, which can be sampled in the urine. Because most of the NADH is formed during ß-oxidation, only 25% of the label enters the TCA cycle and only 10% of the label is expected to be sequestered (6). This estimate is supported by our data, with 88% (SEM = 1.4%) of ²H label recovered when subjects were dosed with d₃-acetate. Hence, ²H-labeled fatty acids have the further advantage of not requiring an individual acetate recovery measurement and can be used independently of an acetate correction factor. An added advantage is that the time taken by the ²H label to metabolize through the intermediate pools of the body is reduced. This is shown by the more rapid appearance of ²H in urine than ¹³C in breath CO₂ after acetate administration. This is probably attributable to the time required by ¹³C to pass through the TCA cycle and internal bicarbonate pools, which is eliminated when ²H label is used, because it mixes very rapidly with the body water pool (16).

Deuterium-labeled fatty acids when oxidized release the label in the body water pool, and sampling of urine measures the abundance of the isotope in the body water pool, whereas measurement of the abundance of ¹³CO₂ in breath is an instantaneous measure of oxidation. Hence, administration of ²H label does not require the frequent measurement of V_CO2 that is needed when using ¹³C label for the calculation of recovery. The use of hydrogen-labeled fatty acids virtually obviates the need for a controlled environment during the collection of a subject's samples. Moreover, because the tracer accumulates in body water, it reduces the potential error of missed peak oxidation with carbon labeling should the peak excretion occur between breath samples, a particularly troublesome issue with rapidly oxidized substrates such as acetate. The use of hydrogen labeling, however, requires the measurement of body water for recovery calculation, correction for water turnover for long recovery times, and is subject to dilution in the large body water pool.

Correction of ²H fatty acid oxidation measures for acetate sequestration did not yield any significant difference against uncorrected data compared with group corrected ¹³C fatty acid oxidation. Because the coefficient of variation for d₃-acetate recovery was only 5% between individuals of all exercise groups, correction using group acetate recovery versus individual recovery did not make a discernible difference in our data. This further emphasizes that deuterium-labeled fatty acids can be used to measure dietary fat oxidation without the need for measurement of acetate correction factor.

In conclusion, we demonstrated that ²H-labeled fatty acids can be used to accurately measure the oxidation of dietary fat during exercise. Minimal sequestration of ²H label in the TCA eliminates the need for an acetate oxidation measure and hence an additional stay in the hospital. Also, compared with ¹³C label, ²H label can be used without the need for frequent sampling, V_CO2 measurement, and restricted environment for measurements. Furthermore, high correlation between ¹³C-labeled fatty acids (corrected for acetate sequestration) and ²H-labeled fatty acids (uncorrected) suggests the accuracy of the measurement of fat oxidation using either method.

	ACKNOWLEDGMENTS

 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30031 and by General Clinical Research Center Grant RR03186. The authors thank the staff at the General Clinical Research Center for their help with inpatient stays and specimen collections.

Manuscript received July 30, 2004 and in revised form August 26, 2004.

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6. Votruba, S. B., S. M. Zeddun, and D. A. Schoeller. 2001. Validation of deuterium labeled fatty acids for the measurement of dietary fat oxidation: a method for measuring fat-oxidation in free-living subjects. Int. J. Obes. Relat. Metab. Disord. 25: 1240–1245.[CrossRef][Medline]

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This Article Abstract Full Text (PDF) All Versions of this Article: M400289-JLR200v1 45/12/2339    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Raman, A. Articles by Schoeller, D. A. Search for Related Content PubMed PubMed Citation Articles by Raman, A. Articles by Schoeller, D. A. Social Bookmarking                      What's this?